Field
This disclosure is concerned generally with the purification of biologically active proteins and specifically with the reduction of Protein A contamination in therapeutic antibody preparations.
Prior Art
Protein A is a well known substance obtained from Staphylococcus aureus, Cowan Strain I. It has a high degree of antibody specificity and the protein has long been used to complex with antibodies. Protein A is commonly used in an immobilized form by being attached to various water insoluble support materials. The immobilized protein A is then used to complex with soluble antibodies which are subsequently eluted from antibody--immobilized protein A complexes. Protein A chromatography is thus an excellent method for purifying ascites or tissue culture fluid derived monoclonal antibodies to homogeneity because of its simplicity and high degree of antibody specificity.
If the purified monoclonal antibodies are intended for therapeutic usage, however, a major safety concern is the possible presence of solubilized Protein A in the purified therapeutic product. Such solubilized Protein A is thought to result from the unintended detachment of Protein A from its support material during the purification process.
Numerous publications link Protein A with toxicity and mitogenicity in animal models and humans (see, for example, Bensinger et al., J. Biol. Resp. Modif. 3, 347, 1984; Messerschmidt et al., J. Biol. Resp. Modif. 3,325, 1984; Terman and Bertram, Eur. J. Cancer Clin. Oncol. 21, 1115; 1985; and Ventura et al., Hortobagyl. Cancer Treat Rep. 71,411, 1987).
Unfortunately, to date there have been no assay methods available to measure very low amounts of Protein A (i.e., less about than 15 pg of Protein A per mg of protein) that might be undesireably present in an antibody preparation intended for therapeutic use. Suprisingly, we found such an assay is now possible. Our assay led to a method of reducing Protein A contamination in antibody preparations to very low levels. The method thus permits the production of therapeutic antibody preparations using immobilized protein A while assuring a low degree of Protein A contamination. Details of our assay and purification methods are described below.